The Rho family GTPases, including Cdc42, Rac, and Rho, were first identified as proteins that have key roles in regulating the organization of the actin cytoskeleton in mammalian fibroblasts (18, 35, 37, 38). Microinjection of activated Cdc42 into fibroblasts causes the induction of filopodia, while activated Rac leads to lamellipodia formation, and activated Rho causes the formation of stress fibers. Both Cdc42Hs and Rac have also been shown to have a role in the dissolution of stress fibers (10, 18, 26), which may reflect an antagonism between these two GTPases and Rho (19, 39).
While they were initially characterized in fibroblasts, the Rho GTPases have also been shown to regulate the morphologies of other types of cells. For example, the Rho GTPases have been shown to have important roles in the regulation of neurite outgrowth in C. elegans, drosophila, chick, and mammalian primary neurons and cell lines (5, 8, 14, 19, 22, 25, 47, 53). This is probably due, at least in part, to the fact that filopodia and lamellipodia play key roles in the elongation of neurites (24). Previous studies have indicated that neurotrophic factors, such as NGF, BDGF, NT-3, NT-4, and chemoattractants such as netrin-1, can induce neurite outgrowth or regulate axon guidance (3, 45).
In mammalian neuronal cell lines, Cdc42 and Rac appear to act antagonistically with Rho. Introduction of constitutively active mutants of Cdc42 and Rac into N1E-115 neuroblastoma cells leads to the formation of neurites (40, 51) and the production of filopodia and lamellipodia in developing growth cones (19), while introduction of dominant negative mutants of Cdc42 and Rac inhibits neurite outgrowth in N1E-115 cells and PC12cells (8, 19, 40, 51). In contrast, activated RhoAV14 causes neurite retraction in PC12 cells and N1E-115 cells, while inhibition of RhoA stimulates the production of neurites in N1E-115 cells (11, 19, 49-51). These results suggest that inactivation of RhoA actually leads to the activation of Cdc42 and Rac, thus leading to the production of neurites (19). Consistent with this, N1E-115 cells form neurites when they are grown in the absence of serum, but not when they are grown in the presence of serum. This is presumably because components in serum, especially LPA, activate Rho, which in turn blocks the production of neurites in response to Cdc42 and Rac (19).
While Cdc42 and Rac clearly have important roles in regulating morphological changes that control growth cone formation and neurite outgrowth, the mechanisms by which the GTPases operate in neuronal cells are still not entirely understood. The identification and characterization of molecular targets for the Rho GTPases is an important step in determining how they control cell morphology in both neuronal cells and non-neuronal cells.
Members of the mammalian p21 activated kinase (“PAK”) family of serine/threonine kinases constitute a family of kinases that bind to Rac and Cdc42, but not Rho (for review see (7, 17, 43)). The PAK family members can be placed into two categories based on their amino acid sequences. The first category includes human PAK1 and PAK2 and mouse PAK3. Each of these protein kinases has a carboxyl terminal kinase domain and an amino terminal regulatory domain. Within the regulatory domain is a GTPase binding domain (“GBD”) that binds to activated Cdc42 and Rac. The regulatory domain also contains two to three proline-rich regions that bind to SH3 domain-containing proteins including the adaptor protein Nck and the exchange factor PIX (7), and a motif that can bind to G protein βγ subunits (7). The members of this family are all quite similar in sequence, exhibiting 73% overall sequence identity and approximately 92% sequence identity within the kinase and GBD domains (43). Members of this subfamily of PAKs are thought to have important roles in regulating cell morphology and cytoskeletal organization, although they may not specifically mediate cytoskeletal effects that are triggered by Cdc42 and Rac. (13, 21, 44, 46).
PAK4 is the first PAK family member to be identified as belonging to a second category of PAKs based on its sequence (1). PAK4 contains an amino terminal GBD and a carboxyl terminal kinase domain, but it does not bind to PIX or Nck and it does not have a G protein βγ-binding motif (1). Furthermore, the GBD and kinase domains of PAK4 have only approximately 50% identity with those of the other PAKs, and the regulatory domain of PAK4 outside of the GBD is completely different from the other PAKs (1). Unlike other PAKs, PAK4 was shown to be a link between Cdc42 and filopodia formation (1). In addition to filopodia formation, PAK4 may also have other functions. For example, a constitutively active PAK4 mutant also leads to the dissolution of stress fibers and focal adhesions, most likely by inhibiting the activity of RhoA (36).